Chinese hamster ovary cells (CHO) are the most widely used cell line for the manufacture of recombinant proteins for pharmaceutical use and processes involving CHO variants account for enormous annual revenue (Andersen and Krummen, 2002). Despite the lack of a fully sequenced genome, a number of important CHO transcriptional profiling studies have been carried out either using non-CHO arrays (Baik et al., 2006) or proprietary CHO cDNA arrays (Wong et al., 2006). These studies have described the effects of both low temperature and the induction of apoptosis during CHO culture. Similarly, a number of proteomic studies have investigated the proteome of CHO and the changes in protein expression in response to culture conditions such as temperature (Baik et al.,2006; Champion et al., 1999; Van Dyk et al. 2003; Kaufmann et al., 1999 Lee et al.,2003). These studies have increased overall understanding of the regulation of CHO function and particularly with respect to the effects of reduced temperature.
Low temperature culture of recombinant production CHO cell lines has been shown to result in sustained viability and increased specific productivity (Al-Fageeh et al., 2006; Fogolin et al., 2004; Furukawa and Ohsuye, 1998; Kaufmann et al., 1999) while maintaining the standard of product quality (Fogolin et al., 2005; Yoon et al., 2003b). The most obvious result of lowering the culture temperature is the immediate reduction in growth rate, other effects include lowered metabolism (glucose consumption, oxygen uptake, lactate & ammonium production) and increased resistance to shear and apoptosis (Chuppa et al., 1997; Furukawa and Ohsuye, 1998; Moore et al., 1997; Yoon et al., 2003a). The reduction in growth rate is linked to an accumulation of cells in G1 phase of the cell cycle (Hendrick et al., 2001; Kaufmann et al., 1999; Yoon et al., 2003 a,b) and G1 phase arrest has been linked to the increased productivity (Fussenegger, 2001).
Due to the reasons listed above, many cell culture processes operate a biphasic culture whereby cells are grown at 37° C. to maximise biomass and then the cells are shifted to a lower temperature to encourage protein production while maintaining a longer and more viable stationary/production phase (Fogolin et al.,2004, 2005; Butler, 2005; Fox et al., 2004). Two of the best-known proteins induced following temperature shift are cold inducible RNA binding protein (CRIP) and RMB3. Of these, CRIP is known to cause growth arrest under conditions of low temperature (Danno et al., 2000; Nishiyama et al., 1997, Sonna et al., 2002) however overall, little is known about how mammalian cells respond to reduced temperatures.
miRNAs are small (˜22nt) non-coding RNAs (ncRNAs) that regulate gene expression at the level of translation. Each miRNA apparently regulates multiple genes and hundreds of miRNA genes are predicted to be present in mammals (Lim et al. 2003). The first miRNA was discovered in C. elegans in 1993 (Lee et al., 1993) and over the last number of years it has become apparent that there are a huge number of these molecules (up to 2% of the human genome encode miRNAs (Miska, 2005)). Recently miRNAs have been found to be critical for development (Ambros, 2003; Chen et al., 2004), cell proliferation and cell death (Brennecke et al. 2003), apoptosis and fat metabolism (Xu et al. 2003), and cell differentiation (Chang et al. 2004).